(1) Field of the Invention
The present invention relates to DNA isolated in a plasmid or in a bacterium encoding a phage resistance protein (designated as AbiE) which aborts infection of the bacterium by the phage. In particular the present invention relates to methods of using the DNA to provide the phage resistance.
(2) Description of Related Art
Lactococcus lactis is widely used in mesophilic milk fermentations to produce cheese, buttermilk, cottage cheese and sour cream. Due to the expanding activities in these industries, there is pressure on L. lactis starter cultures to perform at industrial standards of consistency and efficiency. Phages are the leading cause of fermentation failures during the manufacture of these cultured dairy products (Jarvis A. W., et al., Intervirology 32:2-9 (1991)). They can ruin a fermentation by inactivating the inoculated, sensitive cultures. Since identification of the causal agent in the mid-30s, the dairy industry has learned to manage with this natural phenomenon by developing various solutions such as better sanitation, process modifications and use of phage-resistant cultures (Hill, C., FEMS Microbiol. Rev. 12:87-108 (1993)).
Extensive studies have been carried out on the innate phage resistance mechanisms of L. lactis strains (for review see Hill, C., FEMS Microbiol. Rev. 12:87-108 (1993)). Native barriers against phages have been found and most of them are encoded on plasmids. More than 40 plasmids encoding a variety of phage resistance mechanisms have been identified (Hill, C., FEMS Microbiol. Rev. 12:87-108 (1993)). These anti-phage systems are currently classified in three groups based on their mode of action: blocking of phage adsorption, restriction/modification (R/M), and abortive infection (Abi).
Amongst the natural L. lactis phage resistance mechanisms, the Abi systems are believed to be the most powerful due to their overall effects (Sing, W. D., et al., J. Dairy Sci. 73:2239-2251 (1990)). In a classical abortive infection, the phage lytic cycle is inhibited but after adsorption, DNA injection and early phage gene expression. The end result is the typical Abi.sup.+ phenotype of reduced burst size and plaque size (McLandsborough, L. A., et al., Appl. Environ. Microbiol. 61:2023-2026 (1995); Molineux, I. J., New Biol. 3:230-236 (1991); Sing, W. D., et al., J. Dairy Sci. 73:2239-2251 (1990); Snyder, L., Mol. Microbiol. 15:415-420 (1995)). Generally, the host is also killed in the abortive process. This suicidal outcome traps the phages within the infected cell and limits their dissemination (Sing, W. D., et al., J. Dairy Sci. 73:2239-2251 (1990)).
Many Abi systems have been identified in other bacterial genera (for review see Molineux, I. J., New Biol. 3:230-236 (1991); and Snyder, L., Mol. Microbiol. 15:415-420 (1995)). Some of these systems have been studied extensively but the molecular basis remains somewhat unclear (Snyder, L., Mol. Microbiol. 15:415-420 (1995)). Recent evidence are now only leading to more specific models for their action (Molineux, I. J., New Biol. 3:230-236 (1991); and Snyder, L., Mol. Microbiol. 15:415-420 (1995)). Likewise, Abi systems are very poorly understood in L. lactis (Sing, W. D., et al., J. Dairy Sci. 73:2239-2251 (1990)). To date, five Abi systems have been characterized to molecular level: AbiA (Hill, C., et al., Appl. Environ. Microbiol. 56:2255-2258 (1990), AbiB (Cluzel, P. J., et al., Appl. Environ. Microbiol. 57:3547-3551 (1991), AbiC (Durmaz, E., et al., J. Bacteriol. 174:7463-7469 (1992), AbiD (McLandsborough, L. A., et al., Appl. Environ. Microbiol. 61:2023-2026 (1995) and AbiD1 (Anba, J., et al., J. Bacteriol. 177:3818-3823 (1995). These systems were isolated from different L. lactis strains. There is no homology between the Abi proteins except for AbiD and AbiD1 which shared 28% identity (52% similarity). This was the first indication on the existence of a possible family of Abi proteins in L. lactis (Anba, J., et al., J. Bacteriol. 177:3818-3823). The absence of homology between the other L. lactis Abi mechanisms suggests different mode of action and/or phage targets. Very limited information is available on the molecular mechanisms of L. lactis Abi systems. AbiA is believed to somehow interfere with DNA replication of small isometric phages (Hill, C., et al., Appl. Environ. Microbiol. 57:283-288 (1991); and Moineau, S., et al., Appl. Environ. Microbiol. 59:208-212 (1993)). AbiC does not prevent phage DNA replication but reduces the synthesis of structural phage proteins in infected cells (Durmaz, E., et al., J. Bacteriol. 174:7463-7469 (1992); and Moineau, S., et al., Appl. Environ. Microbiol. 59:208-212 (1993)). Recently, Bidnenko et al. (Bidnenko, E., et al., J. Bacteriol. 177:3824-3829 (1995)) reported that AbiD1 interacted with a small isometric phage operon which contained 4 open reading frames (ORFs). It was proposed that AbiD1 and the orf1 gene product interacted to prevent translation of orf3 RNA (Bidnenko, E., et al., J. Bacteriol. 177:3824-3829 (1995)).
Industrial L. lactis strains with enhanced phage resistance have been constructed by introducing natural plasmids containing Abi systems into phage-sensitive strains (Sanders, M. E., et al., Appl. Environ. Microbiol. 52:1001-1007 (1986)). These improved strains have already been successfully employed for large scale dairy fermentations. However new phages capable of overcoming the introduced Abi defense system have emerged (Alatossava, T., et al., Appl. Environ. Microbiol. 57:1346-1353 (1991); and Moineau, S., et al., Appl. Environ. Microbiol. 59:197-202 (1993)). Thus, the search for novel phage resistance mechanisms is an ongoing objective for culture suppliers (Moineau, S., et al., Appl. Environ. Microbiol. 61:2193-2202 (1995)).
Lactococcal phages are classified in 12 different species based on morphology and DNA homology (Jarvis, A. W., et al., Intervirology 32:2-9 (1991)). Only three have been studied for genetic details because they are commonly encountered worldwide in dairy plants (Jarvis, A. W., et al., Intervirology 32:2-9 (1991)). Members of the species 936 (small isometric heads), c2 (prolate heads) and P335 (small isometric heads) have been, by far, the most disturbing lactococcal phages (Jarvis, A. W., et al., Intervirology 32:2-9 (1991); and Moineau, S., et al., Can. J. Microbiol. 38:875-882 (1992)). DNA-DNA hybridization studies have revealed the absence of significant DNA homology between the three species (Jarvis, A. W., et al., Intervirology 32:2-9 (1991)). The current consensus is that the 936, P335 and c2 species are genetically distinct and L. lactis starter cultures should be resistant against these phages (Moineau, S., et al., Appl. Environ. Microbiol. 61:2193-2202 (1995)).
Objects
It is an object of the present invention to provide DNA encoding a sixth and novel abortive infection mechanism (Abi) from L. lactis which acts prior to or at the phage DNA replication and shares no homology with the previously isolated Abi from L. lactis. Further, it is an object of the present invention to provide a novel Abi system which is efficient against 936, c2 and P335 phages.
Further, it is an object of the present invention to provide a method and bacteria which prevent phage inactivation of Lactococcus lactis strains. Further still, it is an object of the present invention to provide a recombinant bacteria which are economical to prepare and effective in phage inhibition. These and other objects will become increasingly apparent by reference to the following description and the drawings.